Tuberculosis (TB) remains the single most important cause of death among adults worldwide. The high-output pathway of nitric oxide (NO) production is recognized to be important in defense against TB in mice, and new evidence supports it's importance in defense against human TB. The pathway's catalyst, inducible NO synthase (iNOS), is present in alveolar macrophages from patients with TB, and NO-resistant clinical strains of M. tuberculosis (Mtb) appear to disseminate more widely in TB-endemic communities. These recent findings direct our attention to genes in Mtb that may confer resistance to reactive nitrogen intermediates (RNI) alone or along with reactive oxygen intermediates (ROI). During the first grant period, we cloned two such genes, noxR1 and ahpC. noxR1 is novel. ahpC itself is well known, but its role in resistance to RNI is novel. The goals of this grant are to study noxR1 in depth as an RNI-resistance gene and to continue the search for others. The specific aims of the project are as follows: 1) Study the structure and function of noxR1 by site-directed mutagenesis of noxR1, it's expression in E. coli and M. smegmatis, and comparison of their in vitro sensitivity to RNI and ROI and survival in microphages that are wild type (wt), deficient in inducible nitric oxide synthase (inos-I-), deficient in phagocyte oxidase 91-kDa subunit (gp91phox-I-), or deficient in both (PhoxNox-I-); determining NoxR1's mechanism of action and regulation; localizing NoxR1 protein in Mtb; crystallizing recombinant NoxR1 for structural solution by X-ray diffraction. 2) Test the pathophysiologic relevance of NoxR1 by deleting noxR1 in Mtb and testing such derivatives in a murine model of TB; analyzing noxR1 gene sequence and expression in clinical isolates of Mtb varying in RNI- sensitivity. 3) Clone and characterize new RNI/ROI resistance genes by expressing Mtb. Library in E. coli and M. spegmatis; selecting recombinants for survival in wt mice, as well as in the knock-out mice (iNOS, gp91 Phox-I-, and PhoxNOx-I-) and characterizing candidate genes as expressed in E. coli and M. spegmatis for resistance to RNI and ROI in vitro and for survival in macrophages that are wt or deficient in iNOS,gp91phox-I-, or both; studying identified E. coli and M. spegmatis recombinants for survival in vitro in S-nitrosoglutathione, acidified nitrite or peroxynitrite; characterizing candidate genes as expressed in E. coli and M. spegmatis for resistance to RNI and ROI in vitro and for survival in macrophages that are wt or deficient in iNOS,gp91phox, or both. Sequences of the noxR1 region as well as of the newly identified genes will be compared among clinical isolates of Mtb shown to be resistant to RNI and/or ROI in vitro. At the end of 5 years, these studies may yield better understanding of Mtb factors that contribute to resistance to antimicrobial effector molecules naturally produced by human macrophages, and may provide opportunities to discover targets and strategies for new therapies.